Important processes in tissue invasion by blood-borne malignant tumour cells and leukocytes include their adhesion to the luminal surface of the vascular endothelium, their passage through the vascular endothelial cell layer and the subsequent degradation of the underlying basal lamina and extracellular matrix (ECM) with a battery of secreted and/or cell surface protease and glycosidase activities (Nakajima et al, 1983; Schmitt et al, 1992; Vlodavsky et al, 1992). The basal lamina and underlying connective tissue stroma consist predominantly of a complex network of fibronectin, laminin, collagen type IV and vitronectin, each of which interact with heparan sulphate (HS) side chains of heparan sulphate proteoglycans (HSPG) embedded within the matrix (Yurichenco and Schittny, 1990). HS chains generally consist of clusters of sulphated disaccharide units (predominately N-sulphated glucosamine linked 1.fwdarw.4 to .alpha.-L-iduronic acid residues) separated by lowly or non-sulphated regions (predominately disaccharide units of N-acetylated glucosamine linked 1.fwdarw.4 to .beta.-D-glucuronic acid) (Turnbull and Gallagher, 1990,1991). Cleavage of the HS chains by endoglycosidase or heparanase activity produced by invading cells may therefore assist in the disassembly of the ECM and facilitate cell migration. Heparanase activity has been shown to be related to the metastatic potential of murine and human fibrosarcoma and melanoma cell lines (Nakajima et al, 1983, 1986a,b, 1988; Ricoveri and Cappelletti, 1986). Furthermore, heparanase activity has been described in a number of tissues and cell types including rat liver (Gallagher et al, 1988; Hook et al, 1975), human placenta (Klein and Von Figura, 1979; Lider et al, 1989), human platelets (Hoogewerf et al, 1995; Oldberg et al, 1980; Oosta et al, 1982), cultured human skin fibroblasts (Klein and Von Figura, 1976), human neutrophils (Matzner et al, 1985, 1992), activated but not resting rat T-lymphocytes (Naparstek et al, 1984), normal and neoplastic murine B-lymphocytes (Laskov et al, 1991), human monocytes (Sewell et al, 1989) and human umbilical vein endothelial cells (Bartlett et al, 1995; Godder et al, 1991). Because the cleavage of HS appears to be essential for the passage of metastatic tumour cells and leukocytes through basement membranes, studies of heparanase inhibitors provide the potential of developing novel and highly selective classes of anti-metastatic and anti-inflammatory drugs (Coombe et al, 1987; Irimura et al, 1986; Parish et al, 1990; Vlodavsky et al, 1992; Willenborg and Parish 1988).
The expression of heparanase activity by platelets, metastatic tumour cells and circulating cells of the immune system has been related to their involvement in their diapedes and extravasation. Studies have shown that while the initial entrapment of metastatic tumour cells by the capillary endothelium is platelet-independent, platelet aggregation which occurs shortly afterwards can lead to platelet activation and degranulation, resulting in gap formation and retraction of endothelial cells exposing the underlying basement membrane to adhesion by the tumour cells (Tanaka et al, 1986; Crissman et al, 1985; Yahalom et al, 1985). Human platelets have been shown to contain high levels of heparanase activity, capable of degrading endothelial cell surface, tumour-derived and ECM-derived HSPG (Bartlett et al, 1995a, 1995b; Castellot et al, 1982; Hoogewerf et al, 1995; Wasteson et al, 1976, 1977; Yahalom et al, 1984; ) as well as free HS and heparin chains (Graham et al, 1995a and b; Oldberg et al, 1980; Oosta et al, 1982; Wasteson et al, 1976, 1977). To date, three separate mammalian cell heparanase activities have been reported: mouse melanoma B16 heparanase which cleaves HS only, human platelet heparanase which cleaves both heparin and HS and a mouse mastocytoma endoglucuronidase which was reported to cleave newly synthesised heparin precursor but not heparin or HS (Hoogewerf et al 1995; Nakajima et al, 1988; Thunberg et al, 1982). More recent studies have indicated that murine melanoma and macrophage extracts are in fact able to degrade both HS and heparin, however heparin was degraded to a lesser extent than by human platelet extracts (Graham and Underwood, 1996). Although Hennes et al (1988) reported that tumour-derived heparanase was able to degrade matrix HS but was unable to degrade endothelial cell surface HS, human platelets have been shown to degrade endothelial cell surface HS (Wasteson et al, 1977) which was shown by Gamse et al (1978) to be more heparin-like in structure. Thus, it is likely that the platelet heparanase, which is capable of degrading both heparin and HS, may play a critical role in degrading cell-surface HS in focal adhesion plaques, and aiding the extravasation of blood-borne cells.
Following partial purification of human platelet heparanase by Oldberg et al (1980), who determined that the enzyme was an endoglucuronidase which acted upon N-sulphated, iduronic acid-containing heparin biosynthetic intermediates, the enzyme was purified 240,000-fold to apparent homogeneity in 6% yield by a 6-column procedure (Oosta et al, 1982). The enzyme was a 134 kDa single subunit protein which was active towards .sup.125 I-heparin and was confirmed to be an endoglucuronidase. In subsequent studies, the platelet heparanase was shown to cleave endothelial cell surface heparin-like material which inhibited smooth muscle cell proliferation (Castellot et al, 1982) and to cleave the antithrombin III (AT III)-binding octasaccharide between GlcA-GlcNS3S (Thunberg et al, 1982), presumably resulting in the loss of the anticoagulant activity of heparin following its degradation (Oldberg et al, 1980). Although, the purified enzyme was shown to act towards an octasaccharide substrate, platelet extracts were also shown to degrade ECM-derived HS chains to 10 kDa (Yahalom, et al 1984) and to 5 kDa fragments (Freeman and Bartlett, unpublished observations), indicating the existence of specific structural motifs determining the site of cleavage. The size of heparin-cleavage products following platelet heparanase action has not been determined (Graham and Underwood, 1996; Oldberg et al, 1980; Oosta et al, 1982). Recently, however, Hoogewerf et al (1995) reported the 4100-fold purification of a human platelet heparanase activity in 8% yield which was shown to be an endoglucosaminidase that cleaved both heparin and HS principally to disaccharides following radiolabelling of the digestion products. The activity resided in the 8-10 kDa subunit CXC chemokines connective tissue activating peptide-III (CTAP-III) and neutrophil activating peptide-2 (NAP-2) which are members of the platelet basic protein family. In contrast, Graham and Underwood (1996) has since shown that the heparanase activity in human platelet extracts had a Mr of 40-60 kDa, and cleaved both HS and heparin, although the size of the degradation products was not determined.
The resolution of the reported differences in the molecular size (8 to 134 kDa), the substrate specificity (whether the enzyme is an endoglucuronidase or endoglucosaminidase activity), and the size of the substrate cleavage products (disaccharides or 5 to 10 kDa) requires purification of the enzyme(s) to homogeneity in high yield. While some studies of the substrate specificity of the platelet enzyme have been reported (Oldberg, et al 1980; Oosta et al, 1882; Thunberg, et al 1982), surprisingly little has been reported on the inhibition of the enzyme by sulphated polysaccharides (similar to the studies by Nakajima et al (1986a and b)) on the inhibition of tumour cell heparanase activity) apart from the use of some modified heparins and heparin itself to inhibit platelet degradation of ECM-associated HSPG (Eldor, et al 1987). This is especially surprising considering the potentially important role of platelets during the initial stages of tumour cell extravasation.
In order to purify and characterise mammalian heparanase activities and to screen for and develop effective heparanase inhibitors, a simple and rapid assay for heparanase activity is required. However, previous heparanase assays have been cumbersome and time consuming in both preparation of radiolabelled substrate and separation of degradative products from the uncleaved substrate. Frequently heparanase assays have involved the biosynthetic radio-labelling of ECM-associated HSPG and the detection of HS chain degradation by gel filtration analysis of radiolabelled material released from the ECM (Bartlett et al, 1995; Vlodavsky et al, 1992 and references within). Such an approach suffers from the main disadvantage that degradation of HS chains in an ECM involves the synergistic action of proteases which are required to expose the HS chains for subsequent heparanase attack (Bar-Ner et al, 1985, 1986; Benezra et al, 1992; Vlodavsky et al, 1988). Furthermore, most heparanase assays have required extensive degradation of the radiolabelled HS (or ECM-derived HSPG) substrate to allow separation of the degraded product from the substrate by gel filtration (Bartlett et al, 1995; Klein and Von Figura, 1979; Nakajima et al, 1986a; Vlodavsky et al, 1992), although cleavage of HS chains at a single site may be all that is required to allow passage of leukocytes and tumour cells through the basement membrane. Solid phase heparanase assays also have been developed where chemically and biosynthetically radiolabelled heparin and HS chains were attached to a solid support with release of radiolabel from the solid support being a measure of enzyme activity (Nakajima et al, 1986a; Oosta et al, 1982; Sewell et al, 1989). Such assays, however, suffer from the disadvantage that the immobilized substrate may be less accessible to the mammalian heparanase enzyme, and the coupling of the radiolabelled substrate to the solid support, via the substrate's reducing terminus, is a complex and inefficient procedure. Previous studies have also radiolabelled both heparin and HS by iodination at naturally occurring glucosamine residues (Oosta et al, 1982) or by N-acetylation of the partially de-N-sulphated substrate (Nakajima et al, 1986a). Such procedures, however, may result in the masking, or the creation of new heparanase cleavage sites.